Phagocytosis is an important host-mounted defense, used not only to engulf and disable pathogens but also to clear post-apoptotic cells from tissue. Several important human pathogens also survive and replicate intracellularly after entering mammalian cells through phagocytosis. Thus, a detailed understanding of the regulation of phagocytosis may also uncover strategies used by these organisms to establish intracellular infections. Previous studies indicate that phagocytosis of large particles involves Ca2+ dependent exocytosis of the lysosome, and is regulated by the Ca2+ sensor Synaptotagmin VII (Syt VII). These recent results suggest that Syt VII is localized to specialized lysosomal microdomains, which may contain the molecular machinery required for Ca2+-dependent membrane fusion. The Syt Vll-containing microdomains of lysosomal compartments are located proximally to the plasma membrane, consistent with a role in facilitating rapid fusion. In addition, the lysosomal tetraspanin CD63 co-localizes with Syt VII, and may play a structural role in the microdomain organization. To better understand the role of Syt VII localization and function within these microdomains, we propose the following specific aims: 1: Determine the nature of Syt VII association with the tetraspanin CD63 in macrophages. 2: Determine the role of palmitoylation of Syt VII in cellular localization and function. 3: Characterize the SNARE complex involved in Syt Vll-dependent phagocytosis. With these studies, we plan to determine whether Syt VII and CD63 are associated with each other in tetraspanin microdomains, and whether such macrophage microdomains contain specific SNARE molecules responsible for promoting the fusion of lysosomal compartments with nascent phagosomes in response to Ca2+. We will specifically investigate the role of Syt VII palmitoylation in its capacity to associate with CD63 tetraspanin microdomains, using biochemical assays and imaging approaches. Our goal is to determine whether Syt VII palmitoylation regulates the cellular localization of this Ca2+ sensor, and its role in regulating phagocytosis. In additon, these studies will reveal whether CD63 can acts as a chaperone, targeting Syt VII to macrophage lysosomal compartments. The proposed experiments may also identify novel proteins that associate with Syt VII during Ca2+-dependent phagocytosis, which may have a regulatory role in this process.